Mycobacterium avium is responsible for serious opportunistic infections of AIDS patients and other susceptible individuals. M. avium isolates segregate into transparent and opaque colony variants with different pathogenicity and drug susceptibility characteristics. We have identified a new type of phenotypic variation, in which opaque and transparent variants segregate into red- and white-staining subvariants on agar media containing the lipoprotein stain Congo red. White segregants are significantly more resistant to multiple antibiotics than are red segregants. Opaque-transparent variation was previously believed to be the overriding determinant of drug susceptibility in M. avium, but our observations suggest that red-white variation has a greater impact on susceptibility in M. avium, but our observations suggest that red-white variation has a greater impact on susceptibility to some drugs. Using a novel screening approach, we found a genetic polymorphism that co-segregates with the white phenotype. This polymorphism which consists of an open reading frame (ORF1) interrupted by a novel insertion element (IS999), is the first genetic marker of multi-drug resistance to be identified in M. Avium. We propose to 1) determine the role of the ORF::IS999 fusion in red-white variation, 3) characterize the red and white phenotypes in vitro, and 4) determine the impact of red-white variation on disease progression and treatment outcomes in virulence models. Red-white variation is common among clinical isolates of M. avium and a potentially important factor in treatment failure. Understanding of how it occurs could lead to better ways to diagnose and treat multi-drug resistant mycobacterial infections.